CN103484550B - MicroRNA biological markers for early lung cancer diagnosis and application thereof - Google Patents
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Abstract
The invention relates to the field of biological detection, particularly to a group of microRNA biological markers for early lung cancer diagnosis and an application thereof. The microRNA biological marker comprises hsa-miR-125a-5p, hsa-miR-193a-5p, hsa-miR-25, hsa-miR-126 and hsa-miR-34a. The serum miRNA biological markers comprising the 5 miRNAs can not only be particularly applied to early lung cancer detection, but also be applied to advanced lung cancer detection.
Description
Technical field
The present invention relates to field of biological detection, particularly relate to microRNA biomarker and application thereof for one group of lung cancer early diagnosis.
Background technology
Lung cancer is to cause the first cause of whole world cancer mortality, and in the past 3 years, its 5 years survival rates only have slight improvement.Before arriving late period, lung cancer does not have obvious clinical symptom.When making a definite diagnosis over 75% patients with lung cancer, be local late period of lung cancer or pulmonary metastasis, the diagnosis in late period of 3/4ths lung cancer patients is the major causes that cause survival rate low.5 years survival rates of Early stage NSCLC (NSCLC) patient are approximately 50%-70%, and by contrast, III phase and IV phase NSCLC patient's survival rate is respectively 5%-15% and lower than 2%.Early diagnosis patients with lung cancer can effectively reduce its mortality ratio.Chest X-ray and phlegm cytology checking are for detection of some early stage of lung cancer, but sensitivity is low.Although the detection that computed tomography (CT) can the early stage small volume NSCLC of non-invasive detection, it has higher false positive rate.In addition, CT scan may make patient be exposed among harmful radiation, causes more tumour to occur.Several protein markers, as cytokeratin 19 fragment (CYFRA21-1), carcinomebryonic antigen (CEA), neuronspecific enolase (NSE) etc. is used for lung cancer to carry out without surgical diagnosis, but the sensitivity of these marks and specificity are not high.Therefore, need to find Novel noninvasive, highly sensitive and specific mark lung cancer is carried out to early detection.
MicroRNAs (miRNAs) is that a class is little, the noncoding RNA of high conservative, and they have effect in multiple evolution, and can be used as and transcribe rear regulatory factor regulatory gene and express.They regulate regulate several biological processes, comprise differentiation, proliferation and apoptosis.The change of miRNA expression level is relevant with kinds cancer, and they are as oncogene or tumor suppressor gene.It is reported, miRNA exists in serum human with a kind of high stability form, can resist digestion and the exacting terms of RNA enzyme.Because serum ratio is easier to obtain, circulating biological mark is hopeful the detection for cancer as the mark of non-intrusion type.For the mark that develops non-intrusion type is for early detection lung cancer, we assess the expression level of the serum miRNAs in early stage of lung cancer patient and normal healthy controls crowd, verify that miRNA can be used as biomarker and distinguishes this two groups of crowds.
Lung tumor is a kind of different substantiality disease, by complicated and multiple process, is developed.Therefore, we infer, the serum miRNA that simultaneously assesses a plurality of Tumor-assaciateds can provide an extremely sensitive and special diagnostic test for the early stage of lung cancer.In order to verify this hypothesis, we have assessed the expression level of 5 miRNAs in early stage of lung cancer patient, and assess them for the diagnostic value of the early stage of lung cancer.
Summary of the invention
The shortcoming of prior art in view of the above, the object of the present invention is to provide microRNA biomarker and application thereof for one group of lung cancer early diagnosis, and its relevant miRNA probe and diagnostic kit is further provided, for solving the problems of the prior art.
For achieving the above object and other relevant objects, first aspect present invention provides one group of pulmonary cancer diagnosis miRNA biomarker, described miRNA biomarker comprises: hsa-miR-125a-5p(MIMAT0000443), hsa-miR-193a-5p(MIMAT0004614), hsa-miR-25(MIMAT0000081), hsa-miR-126(MIMAT0000445) and hsa-miR-34a(MIMAT0000255).
The sequence of described hsa-miR-125a-5p is as shown in table 1Seq ID No.1;
The sequence of described hsa-miR-193a-5p is as shown in table 1Seq ID No.2;
The sequence of described hsa-miR-25 is as shown in table 1Seq ID No.3;
The sequence of described hsa-miR-126 is as shown in table 1Seq ID No.4;
The sequence of described hsa-miR-34a is as shown in table 1Seq ID No.5.
Preferably, described miRNA is combined as serum miRNA biomarker.
Serum miRNA biomarker of the present invention, joins together to comprise I-IV phase people lung cancer for the detection of people's lung cancer, be particularly suitable for the detection of early stage people's lung cancer, described early stage people's lung cancer specifically refers to I/II phase people lung cancer.
Second aspect present invention provides the purposes of described miRNA biomarker in the diagnosing tumor medicine of preparation or screening people lung cancer.
Preferably, described miRNA biomarker is in preparation or screen the purposes in the diagnosing tumor medicine of early stage people's lung cancer.
Third aspect present invention provides the combination of miRNA primer, probe for one group of lung cancer early diagnosis, the combination of described miRNA primer, probe comprises: hsa-miR-125a-5p primer, probe, hsa-miR-193a-5p primer, probe, hsa-miR-25 primer, probe, hsa-miR-126 primer, probe, hsa-miR-34a primer, probe.
Described each miRNA primer specifically comprises primer after primer before the reverse transcription primer, quantitative PCR of each miRNA, quantitative PCR.
Preferably, the reverse transcription primer sequence of described hsa-miR-125a-5p as described in Seq ID No.7, before quantitative PCR primer sequence as described in Seq ID No.13, after quantitative PCR primer sequence as described in Seq ID No.19.
Preferably, the reverse transcription primer sequence of described hsa-miR-193a-5p as described in Seq ID No.8, before quantitative PCR primer sequence as described in Seq ID No.14, after quantitative PCR primer sequence as described in Seq ID No.19.
Preferably, the reverse transcription primer sequence of described hsa-miR-25 as described in Seq ID No.9, before quantitative PCR primer sequence as described in Seq ID No.15, after quantitative PCR primer sequence as described in Seq ID No.19.
Preferably, the reverse transcription primer sequence of described hsa-miR-126 as described in Seq ID No.10, before quantitative PCR primer sequence as described in Seq ID No.16, after quantitative PCR primer sequence as described in Seq ID No.19.
Preferably, the reverse transcription primer sequence of described hsa-miR-34a as described in Seq ID No.11, before quantitative PCR primer sequence as described in Seq ID No.17, after quantitative PCR primer sequence as described in Seq ID No.19.
Preferably, the probe sequence of described each miRNA probe is as shown in Seq ID No.20.
Fourth aspect present invention provides the purposes in the diagnosing tumor medicine that is combined in preparation or screening people lung cancer of described miRNA primer, probe.
Fifth aspect present invention provides a kind of tumor diagnosis kit of people's lung cancer, comprises the combination of described miRNA primer, probe.
Preferably, described test kit also comprises Taq enzyme, dNTP, MgCl
2with PCR damping fluid.
Inventor is by studies confirm that, early stage of lung cancer patient's Sera of Lung Cancer is compared with normal control, hsa-miR-125a-5p, hsa-miR-193a-5p, hsa-miR-25 and hsa-miR-126 lower and express, hsa-miR-34a up-regulated expression (p<0.0001), the expression level of 5 miRNAs changes 0.23-2.55 doubly (table 2).The AUC that these 5 miRNAs join together can reach 0.966, and sensitivity and specificity are respectively 92.3% and 90.7%.
Further, inventor also confirms by research, in the Sera of Lung Cancer of III/IV phase lung cancer patient, hsa-miR-125a-5p, hsa-miR-193a-5p, hsa-miR-25 and hsa-miR-126 lower and express, hsa-miR-34a up-regulated expression (p<0.001), and the expression level there was no significant difference of these 5 miRNAs in III/IV phase lung cancer patient and in I/II phase patient, meanwhile, the also there was no significant difference (p>0.05) of miRNAs expression level between two groups of contrasts.The AUC that 5 miRNAs distinguish III/IV phase lung cancer patient and normal control is 0.710-0.911, and sensitivity is 66.7%-96.7%, and specificity is 70.0%-87.5%.The AUC that these 5 miRNAs join together to distinguish III/IV lung cancer patient and normal control is 0.972, sensitivity and specificity are respectively 91.7% and 90.7%, compare there was no significant difference (p>0.05) aspect sensitivity and specificity with detecting the early stage of lung cancer.
Visible, the serum miRNA biomarker that these 5 miRNAs provided by the present invention form not only can be specially adapted to detection of early lung cancer, can also be for the detection of advanced lung cancer.In addition, lung cancer patient is carried out to layering comparison according to age, sex and smoking history, show these clinical factors all with the expression level of miRNAs irrelevant (p>0.05).Therefore, these 5 miRNAs join together for the detection in early stage and late period of lung cancer, especially to the early stage of lung cancer, to have very high diagnostic value.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of probe method quantitative PCR detection miRNA.
The expression level that Fig. 2 A-M is shown as 13 miRNAs in preliminary screening changes schematic diagram (due to hsa-miR-200b, the Cp value of hsa-miR-34c and hsa-miR-34c-3p is greater than 36, so will not add up).
Fig. 3 A-E is shown as the expression level of 5 miRNAs of the present invention in 52 routine early stage of lung cancer serum and 54 routine normal control serum and changes schematic diagram (all p<0.0001).
Fig. 4 A-E is shown as 5 miRNAs of the present invention for distinguishing the ROC graphic representation of early stage of lung cancer sample and normal control.Fig. 4 F is shown as the ROC graphic representation that 4 protein markers and 5 serum miRNAs distinguish early stage of lung cancer sample and normal control, dotted line represents the ROC graphic representation of 4 protein marker joint-detection early stages of lung cancer, and solid line represents the ROC graphic representation of 5 miRNAs joint-detection early stages of lung cancer.
Embodiment
Below, by specific specific examples explanation embodiments of the present invention, those skilled in the art can understand other advantages of the present invention and effect easily by the disclosed content of this specification sheets.The present invention can also be implemented or be applied by other different embodiment, and the every details in this specification sheets also can be based on different viewpoints and application, carries out various modifications or change not deviating under spirit of the present invention.
Before further describing the specific embodiment of the invention, should be understood that protection scope of the present invention is not limited to following specific specific embodiments; It is also understood that the term using in the embodiment of the present invention is in order to describe specific specific embodiments, rather than in order to limit the scope of the invention; In specification sheets of the present invention and claims, unless explicitly pointed out in addition in literary composition, singulative " ", " one " and " this " comprise plural form.
When embodiment provides numerical range, unless should be understood that the present invention is otherwise noted, between two end points of each numerical range and two end points, any one numerical value all can be selected.Unless otherwise defined, the same meaning that all technology of using in the present invention and scientific terminology and those skilled in the art of the present technique understand conventionally.The concrete grammar using in embodiment, equipment, material, according to those skilled in the art to the grasp of prior art and record of the present invention, can also with to the method described in the embodiment of the present invention, equipment, material is similar or any method, equipment and the material of the prior art that is equal to are realized the present invention.
Unless otherwise indicated, in the present invention, disclosed experimental technique, detection method, preparation method all adopt the routine techniques of molecular biology, biological chemistry, chromatin Structure and analysis, analytical chemistry, cell cultures, recombinant DNA technology and the association area of the art routine.These technology are existing perfect explanation in existing document, specifically can be referring to MOLECULAR CLONING:A LABORATORY MANUAL such as Sambrook, Second edition, Cold Spring Harbor Laboratory Press, 1989and Third edition, 2001; Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; The series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc.
Lung cancer sample in the present invention is collected by upper marine mountain hospital.When getting blood, collect each patient's clinical information simultaneously.The morphology of tumour is determined according to WHO standard, according to the 7th edition TNM classification of malignant tumors standard, these patients is carried out by stages.All patients' blood is all to collect in pulmonary cancer diagnosis, prior to tumor resection or treatment.Normal healthy controls sample is taken at Shanghai Ruijin Hospital, and the negative findings detecting according to blood count, chest X-ray and abdominal ultrasonic is collected.The serum of collecting is stored in-80 ℃ at once, until use.
The extracting of serum RNA and quantitative
Use mirVana miRNA extraction agent box, according to specification sheets extracted total RNA from 200 μ L serum, final elution volume is 100 μ L.
Because RNA concentration is lower, we use fixing volume for follow-up reverse transcription reaction.Reverse transcription reaction is used Taqman MicroRNA reverse transcription test kit, according to specification sheets, operates.Taqman PCR test kit is used in quantitative PCR reaction, and instrument is LightCycler480.All quantitative PCR reactions are all provided with three multiple holes, and every secondary response is all provided with negative control.We use the RNA(cel-miRNA-39 of one section of nematode, MIMAT0000010, Seq ID No.6) as quantitative internal reference.
The selection of serum miRNA biomarker
We use quantitative PCR (24 routine early stage of lung cancer samples and 24 routine normal controls) in a collection of serum sample to detect 16 miRNAs(has-miR-21, has-miR-155, has-miR-205, has-miR-20a, hsa-miR-200b, hsa-miR-375, hsa-miR-486-5p, hsa-miR-186, hsa-miR-186-3p, hsa-miR-34c, hsa-miR-34c-3p, hsa-miR-126, hsa-miR-34a, hsa-miR-125a-5p, hsa-miR-193a-5p and hsa-miR-25) expression.There is the miRNA of significance differential expression further with sample, to verify its expression.
Data statistic analysis
Use GraphPad Prism5.01 and SPSS17.0 to carry out statistical study to data.Data represent with the form of mean value ± standard error, unless there is special explanation.Serum miRNA expression level presents with the multiple version with respect to normal control.Mann-Whitney U check is for comparing the differential expression of lung cancer patient group and Normal group serum miRNA.All p values are bilateral, and p<0.05 has been considered to statistical significance.
Embodiment 1
Initial option mark in small portion serum sample
First we detect 16 miRNAs (has-miR-21, has-miR-155, has-miR-205 in 48 routine serum (the early stage patient of 24 routine lung cancer and 24 routine normal controls), has-miR-20a, hsa-miR-200b, hsa-miR-375, hsa-miR-486-5p, hsa-miR-186, hsa-miR-186-3p, hsa-miR-34c, hsa-miR-34c-3p, hsa-miR-126, hsa-miR-34a, hsa-miR-125a-5p, hsa-miR-193a-5p and hsa-miR-25) expression.Result shows, compares with normal control, and in Sera of Lung Cancer, hsa-miR-125a-5p, hsa-miR-193a-5p, hsa-miR-25 and hsa-miR-126 lower and express, hsa-miR-34a up-regulated expression (p<0.001).Hsa-miR-200b, hsa-miR-34c and hsa-miR-34c-3p expression level in serum too low (Cp>36), can not carry out accurate quantitative analysis.Has-miR-21, has-miR-155, has-miR-205, has-miR-20a, hsa-miR-375, hsa-miR-486-5p, hsa-miR-186-3p and hsa-miR-186 two groups of expression in lung cancer early stage blood serum sample and normal serum sample do not have significant difference (p>0.05) (Fig. 2).
Embodiment 2
The further checking of miRNA mark
In order to verify preliminary screening 5 miRNAs(hsa-miR-125a-5p out, hsa-miR-193a-5p, hsa-miR-25, hsa-miR-126 and hsa-miR-34a) diagnostic value, the further expression level of these 5 miRNAs of checking in 106 routine serum samples.48 routine samples in preliminary screening step are also contained in the statistical study of verification step (106 routine serum samples herein comprise 52 routine I/II phase lung cancer patient serum samples and 54 routine normal control serum samples).
As shown in Figure 1, during each miRNA quantitative PCR, the primer and probe are as follows for the principle of probe method quantitative PCR detection miRNA:
Table 1.miRNA sequence, reverse transcription primer, the front primer of quantitative PCR, general rear primer and probe sequence
Note: underscore flag sequence is and #21 probe identical sequence.
Result shows, normal control is compared, in Sera of Lung Cancer, and hsa-miR-125a-5p, hsa-miR-193a-5p, hsa-miR-25 and hsa-miR-126 lower and express, hsa-miR-34a up-regulated expression (p<0.0001, Fig. 3).Compare with normal control, in the early stage patients serum of lung cancer, the expression level of 5 miRNAs changes 0.23-2.55 doubly (as shown in table 2).These results show that serum miRNAs can be used as the biomarker of lung cancer early diagnosis.
The expression of results of table 2.5 miRNAs in 52 routine early stage of lung cancer serum and 54 routine normal control serum
Embodiment 3
Experimenter's operating characteristic (ROC) tracing analysis
Building ROC curve comes 5 serum miRNAs of comparison to distinguish the diagnosis capability of early stage of lung cancer patient and normal healthy controls.The area under curve of 5 miRNAs ROC (AUC) is as follows respectively: hsa-miR-125a-5p, 0.840 (95% fiducial interval, 0.760-0.920); Hsa-miR-193a-5p, 0.819 (95% fiducial interval, 0.736-0.902); Hsa-miR-25,0.814 (95% fiducial interval, 0.734-0.894); Hsa-miR-126,0.843 (95% fiducial interval, 0.765-0.920); Hsa-miR-34a, 0.738 (95% fiducial interval, 0.644-0.832) (Fig. 4 A-4E).Under best cutoff value, sensitivity and the specificity of miRNAs are as follows: hsa-miR-125a-5p, is respectively 83.3% and 82.7%; Hsa-miR-193a, 64.8% and 94.2%; Hsa-miR-25,90.7% and 61.5%; Hsa-miR-126,70.4% and 82.5%; Hsa-miR-34a, 67.3% and 74.1%.The AUC that these 5 miRNAs join together can reach 0.966, sensitivity and specificity be respectively 92.3% and 90.7%(as shown in Fig. 4 F).We have also assessed CYFRA21-1, CEA, and tetra-protein markers of NSE and SCC-Ag join together to detect early stage of lung cancer ability.The AUC that these 4 protein markers join together to distinguish the early stage of lung cancer and normal people can reach 0.928(95% fiducial interval, 0.882-0.975), sensitivity and specificity be respectively 83.3% and 88.5%(Fig. 4 F).These results show, the lung cancer protein marker conventional with these compared, and 5 miRNAs combine detection of early lung cancer is had to higher sensitivity and specificity.
Embodiment 4
In III/IV phase lung cancer patient, further verify the diagnosis capability of 5 serum miRNAs
In order further to verify the diagnosis capability of 5 serum miRNAs, we detect the expression of 5 serum miRNAs in 30 routine normal controls and 48 routine III/IV phase lung cancer patients.Compare with normal control, in Sera of Lung Cancer, miR-125a-5p, miR-193a-5p, miR-25 and miR-126 lower and express, miR-34a up-regulated expression (p<0.001).The expression level there was no significant difference of these 5 miRNAs in III/IV phase lung cancer patient and in I/II phase patient, meanwhile, the miRNAs expression level between two groups of contrasts is there was no significant difference (p>0.05) also.The AUC that 5 miRNAs distinguish III/IV phase lung cancer patient and normal control is 0.710-0.911, and sensitivity is 66.7%-96.7%, and specificity is that 70.0%-87.5%(is as shown in table 3).
The expression of results of table 3.5 miRNAs in 48 routine III/IV phase Sera of Lung Cancers and 30 routine normal control serum
The AUC that these 5 miRNAs join together to distinguish III/IV lung cancer patient and normal control is 0.972, sensitivity and specificity are respectively 91.7% and 90.7%, compare there was no significant difference (p>0.05) aspect sensitivity and specificity with detecting the early stage of lung cancer.In addition, by lung cancer patient according to age, sex and smoking history carry out layering relatively show these clinical factors all with irrelevant (p>0.05) (as shown in table 4) of the expression level of miRNAs.These results show that these 5 miRNAs not only can be for detection of early lung cancer, can also be for the detection of advanced lung cancer.
Table 4. lung cancer sample carries out the p value result of layer analysis according to age, sex and smoking history
| ? | miR-125a-5p | miR-193a-5p | miR-25 | miR-126 | miR-34a |
| The I/II phase | ? | ? | ? | ? | ? |
| Age (> 60vs≤60) | 0.803571693 | 0.333407567 | 0.651705552 | 0.187726783 | 0.444506021 |
| Sex (male vs female) | 0.674771171 | 0.7161111216 | 0.081209989 | 0.569489701 | 0.962813531 |
| Smoker vs non-smoker | 0.48154138 | 0.766965009 | 0.604034512 | 0.159235091 | 0.436621379 |
| The III/IV phase | ? | ? | ? | ? | ? |
| Age (> 60vs≤60) | 0.263845700 | 0.178639596 | 0.068627704 | 0.106539002 | 0.717267410 |
| Sex (male vs female) | 0.116853293 | 0.585788415 | 0.815334594 | 0.178402180 | 0.632483837 |
| Smoker vs non-smoker | 0.476467048 | 0.061146231 | 0.140054905 | 0.438984574 | 0.145672442 |
In sum, the present invention has effectively overcome various shortcoming of the prior art and tool high industrial utilization.
Above-described embodiment is illustrative principle of the present invention and effect thereof only, but not for limiting the present invention.Any person skilled in the art scholar all can, under spirit of the present invention and category, modify or change above-described embodiment.Therefore, such as in affiliated technical field, have and conventionally know that the knowledgeable, not departing from all equivalence modifications that complete under disclosed spirit and technological thought or changing, must be contained by claim of the present invention.
Claims (7)
1. one group of lung cancer early diagnosis miRNA biomarker, described miRNA biomarker is comprised of following miRNA: hsa-miR-125a-5p, hsa-miR-193a-5p, hsa-miR-25, hsa-miR-126 and hsa-miR-34a, described miRNA biomarker is serum miRNA biomarker.
2. the purposes of miRNA biomarker as claimed in claim 1 in the diagnosing tumor medicine of screening people lung cancer.
3. the combination of miRNA primer, probe for one group of pulmonary cancer diagnosis, the combination of described miRNA primer, probe comprises: hsa-miR-125a-5p primer, probe, hsa-miR-193a-5p primer, probe, hsa-miR-25 primer, probe, hsa-miR-126 primer, probe, hsa-miR-34a primer, probe.
4. the combination of miRNA primer, probe for one group of pulmonary cancer diagnosis as claimed in claim 3, is characterized in that, described each miRNA primer specifically comprises primer after primer before the reverse transcription primer, quantitative PCR of each miRNA, quantitative PCR.
5. the combination of one group of pulmonary cancer diagnosis miRNA primer as claimed in claim 4, probe, it is characterized in that, the reverse transcription primer sequence of described hsa-miR-125a-5p as described in Seq ID No.7, before quantitative PCR primer sequence as described in Seq ID No.13, after quantitative PCR primer sequence as described in Seq ID No.19; The reverse transcription primer sequence of described hsa-miR-193a-5p as described in Seq ID No.8, before quantitative PCR primer sequence as described in Seq ID No.14, after quantitative PCR primer sequence as described in Seq ID No.19; The reverse transcription primer sequence of described hsa-miR-25 as described in Seq ID No.9, before quantitative PCR primer sequence as described in Seq ID No.15, after quantitative PCR primer sequence as described in Seq ID No.19; The reverse transcription primer sequence of described hsa-miR-126 as described in Seq ID No.10, before quantitative PCR primer sequence as described in Seq ID No.16, after quantitative PCR primer sequence as described in Seq ID No.19; The reverse transcription primer sequence of described hsa-miR-34a as described in Seq ID No.11, before quantitative PCR primer sequence as described in Seq ID No.17, after quantitative PCR primer sequence as described in Seq ID No.19; The probe sequence of described each miRNA probe is as shown in Seq ID No.20.
6. the purposes in the diagnosing tumor medicine that is combined in preparation or screening people lung cancer of the miRNA primer as described in claim as arbitrary in claim 3-5, probe.
7. a tumor diagnosis kit for people's lung cancer, comprises the miRNA primer described in the arbitrary claim of claim 3-5, the combination of probe.
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Inventor after: Mao Hongju Inventor after: Wang Ping Inventor after: Zhang Honglian Inventor after: Yang Dawei Inventor after: Hong Qunying Inventor after: Jin Qinghui Inventor after: Bai Chunxue Inventor after: Zhao Jianlong Inventor before: Mao Hongju Inventor before: Wang Ping Inventor before: Zhang Honglian Inventor before: Yang Dawei Inventor before: Hong Qunying Inventor before: Jin Qinghui Inventor before: Bai Chunxue Inventor before: Zhao Jianlong |